Pseudomonas aeruginosa is an opportunistic pathogen responsible for both acute and persistent infections in individuals with cystic fibrosis (CF), burn victims, ventilator patients, and those who have had intestinal reconstruction. P. aeruginosa is among the most common nosocomial pathogens for intensive care unit patients. For P. aeruginosa and many bacterial pathogens, the most persistent of these infections, and the hardest to treat with antibiotics, are those that form bacterial biofilms. Often, a first step n establishing biofilms from the planktonic state is swarming, where bacteria use surface motility to colonize new surfaces in groups. Previous reports for P. aeruginosa swarming have shown importance of production of a surfactant called rhamnolipid, but it was recently determined that rhamnolipid production is not required for swarming when growing with glutamate. Such a glutamate-dependent phenotype has obvious links to pathogenesis, particularly for lung infections. For example, amino acid auxotrophs are routinely isolated from lung sputum of CF patients. A transposon mutagenesis approach will be used to determine the genes that confer the glutamate-effect and then systematically characterize and confirm the importance of the random mutants that show a loss of swarming on glutamate. The molecular nature, regulation, and activity of the surfactant produced by P. aeruginosa when growing on glutamate will be probed using proteomics, wet chemistry, and analytical chemistry protocols to characterize this surfactant and the protein(s) required for its synthesis. The broader activity of this surfactant wll be examined using biofilm and cytotoxicity assays. Using the clean deletion mutants and chemical data, the importance of this glutamate-dependent swarm phenotype will be explored on various media including synthetic CF sputum. Discerning how amino acids like glutamate dictate swarm motility and biofilm development for P. aeruginosa may greatly improve our understanding of pathogenesis. This knowledge may be important to control and prevent many respiratory and nosocomial biofilm infections. Completion of this research will provide new information about a regulatory response of P. aeruginosa to the amino acid glutamate and amino acids-dependent phenotypes that have been strongly associated with infection for P. aeruginosa and other pathogens. This will improve our understanding of bacterial behavior at the onset of infection. Additionally, because many virulence activities of P. aeruginosa are associated with AHL quorum sensing, it will be important to explain this glutamate-dependent phenotype where AHL quorum sensing appears to not be required.